Various arrays of polynucleotides (such as RNA and DNA) are known and used in genetic testing, screening and diagnostics. Arrays are defined by the regions of different biopolymers or nucleotides arranged in a predetermined configuration on a substrate. Most importantly, the arrays when exposed to a population of analytes will exhibit a pattern indicative of the presence of the various components separated spatially. Array binding patterns of polynucleotides and/or peptides can be detected by using a variety of suitable target labels. Once bound to the array, these target labels can then be quantified and observed and the overall pattern on the array determined.
A number of methods have been designed for manufacturing micro arrays. DNA micro arrays are particularly useful for analyzing large sets of genes through “gene expression profiling”. Using various techniques, arrays can be used to effectively analyze genomes and portions of genomes. Probe arrays have been produced by a variety of means. However, two major methods exist for fabricating arrays used in expression profiling. The first technique uses chemical methods to synthesize polynucleotide probes in situ on array surfaces. This technique uses addressable adaptions of phosphoramidite chemistry. In the second method, polynucleotide probes synthesized enzymatically or chemically can be deposited and attached to a surface through covalent or non-covalent means. The enzymatic method is particularly effective in fabricating arrays with larger probes of (100–1000 nucleotides).
A number of steps are used in the fabrication of the micro arrays designed in the in situ process. The first step in the in situ process is to deposit a polymeric layer on top of a glass substrate or similar type material. Once the polymeric layer has been deposited phosphoramidite chemistry is used to build the oligonucleotides on the micro arrays in a step-wise fashion. This is accomplished by adding one monomer at a time until the final polynucleotide is constructed. The steps of construction using these methodologies are well known in the art and generally include a coupling step followed by a series of optionally capping, oxidation and deblocking steps. The final constructed oligonucleotide can then be employed for binding targets of known or unknown sequences.
Other methods are known in the art that can also be used for fabricating micro arrays. For instance, oligonucleotides or oligonucleotide fragments have been deposited directly on polymer surfaces. After the deposition process the deposited oligonucleotides are then subjected to a drying step and a final curing step. The curing step includes the application of heat, UV light or other similar physical or chemical methods to cross-link the polynucleotides to the surface. Processes have also been designed in which cDNA is used in place of polynucleotides and their fragments.
The above methods have been employed for constructing micro arrays in various sizes and designs. The utilization process, however, provides a number of problems (i.e hybridization) related to reading the signals that are produced from the probes on the array surface. The utilization process provides a variety of potential contaminants that may provide non-specific binding of molecules that could affect the overall final readings or signals produced from the arrays. For instance, a number of dyes and blocking or staining agents are used in the hybridization process. Some of the dyes or agents may or may not be attached to the target sample to be analyzed, and may provide for a source of contamination by sticking to the probes or substrate surfaces. It is not clear what the source of these contaminants might be. However, it is likely that the binding mechanism to the probes and surfaces for these contaminants might be by Van der Waals interactions and hydrogen bonding. Also, it is know that these contaminants not only interfere or effect the final fluorescence signal produced by the probe bound to the target, but also the background signal inside the feature areas.
A number of techniques have been developed to deal with this issue of background noise and contamination. Threshold levels can be set so that only higher signals can be read by a scanner or detector and the probe to target binding determined. Other techniques include using areas of low fluorescence, typically outside of the feature locations to use a calibration point for background determinations. This technique requires one to assume that the areas of lowest fluorescence represent areas of non-bound probe to target. Areas with highest fluorescence or signal are due to the probe to target molecules that contain a fluorescent tag. The most important problem with this technique is that these areas may have been bound or affected by a fluorescent contaminant or some other stray contaminant that interferes with the fluorescent signal that is produced. It would be desirable, therefore, to have a way for determining accurate background signal so that the actual signal produced from a probe/target molecules can be determined.
These and other problems with the prior art processes and designs are obviated by the present invention. The references cited in this application infra and supra, are hereby incorporated in this application by reference. However, cited references or art are not admitted to be prior art to this application.